Seroprevalence and risk factors for bovine brucellosis in domestic yaks (Bos grunniens) in Tibet, China.

A cross-sectional study was conducted in three counties (Damxung, Maizhokunggar and Yadong) in Tibet in April and May 2015. A total of 1,523 yaks owned by 181 herders were randomly selected and blood sampled. Sera were tested using the rose bengal test (RBT) and a competitive immune-enzymatic assay (C-ELISA) and the test results interpreted in parallel. The individual yak prevalence was 2.8% (95% CI 2.0-3.7) with a herd prevalence of 18.2% (95% CI 12.9-24.6). At the individual level, two predictor variables, age and production system, were significantly associated with seropositivity by a binary logistic regression analysis. The odds of Brucella infection were significantly higher in older Yaks (3-5 years old, OR = 4.51; 95% CI 1.53-19.29; ≥6 years old, OR = 3.89; 95% CI 1.23-17.21) compared to those of younger yaks (≤2 years old). The odds of seropositivity for yaks managed under an agro-pastoral production system were 2.9 (95% CI 1.48-5.86) times higher compared to those managed under a pastoral production system. At the herd level, an association between the infection with Brucella and a history of abortions in the herd was observed (OR = 4.98, 95% CI 1.48-16.62). Surprisingly, vaccination was not associated with a lower level of infection (p = 0.49 and p = 0.99 for individual and herd level data, respectively). The results of the survey indicate that bovine brucellosis is endemic among the yak population in the plateau region of China, and the risk factors identified in the study should be considered in the epidemiology of the disease and when developing control programs for the disease.

Development of an epitope-based competitive ELISA for the detection of antibodies against Tibetan peste des petits ruminants virus.

AIMS: To develop an effective diagnostic kit, based on a competitive ELISA-based system (cELISA), for detecting serum antibody against peste des petits ruminants virus (PPRV). METHODS: Epitope peptides of the nucleocapsid (N) protein of Tibetan PPRV were synthesized chemically and injected into rabbits to prepare hyperimmune antisera. Test sera were incubated simultaneously with hyperimmune antisera and added to the wells of ELISA plates coated previously with recombinant N protein. Horseradish peroxidase-conjugated goat anti-rabbit antibody was employed to detect the quantity of hyperimmune antisera combined with recombinant N protein. RESULTS: A cELISA has been developed for monitoring PPRV infections with a cutoff value of 35. Relative sensitivity and specificity values of the epitope-based cELISA were 96.18 and 91.29%, respectively, when compared with a commercial cELISA kit in a test involving 1,039 serum samples. CONCLUSION: We report an efficient method for preparing antibody suitable for incorporation into a cELISA that can be used routinely for the detection of PPRV antibodies in serum samples. The method eliminated the requirement for virus culture and monoclonal antibody preparation, reduced the biorisk posed by virus-dependent manipulations, and the performance of the resultant cELISA compared favorably with a commercially available cELISA kit.

Development of an indirect ELISA with artificially synthesized N protein of PPR virus.

The full-length gene encoding the nucleocapsid (N) protein of the virus (PPRV) responsible for an outbreak of peste des petits ruminants in Tibet in 2007 was synthesized in two stages using overlapping PCR without the need for viral genomic cDNA as template. The full-length N gene was successfully expressed in Escherichia coli, and the purified gene product bound to monoclonal antibody raised against PPRV N protein. Furthermore, it was able to replace recombinant B-N antigen as the coating antigen in a commercial ELISA kit prepared with another PPRV strain. Recombinant protein was employed as the coating antigen to develop an indirect ELISA for PPRV antibody detection in the sera of infected small ruminants. Antibody detection was optimal at a 1:200 serum dilution and an antigen concentration of 3.2 µg/ml, and the positive threshold (cutoff) value of the assay was 2.18. Analysis of 697 serum samples revealed the sensitivity and specificity of the indirect ELISA to be 96.7 and 96.1%, respectively, compared with a commercially available ELISA test.